*2.11. Western Blotting of Promastigote Cell Lysate*

Preparation of the parasite lysate and Western blot analysis was performed following the method described earlier [58]. K133WT and K133AS-R cell lysates (100 µG) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membrane strips were blocked and incubated sequentially with anti-AQP1 (1:1000), anti-HSP70 (1:500), or anti-tubulin (1:1000) (endogenous control) primary antibodies. Following this, the membrane was probed with Horse radish Peroxidase (HRP)-conjugated anti-rabbit IgG (1:80,000) produced in mice (Sigma Aldrich, St. Louis, MO, USA). Blot was developed using Western blot detection enhanced chemiluminescence (ECL) detection reagent (Merck, Burlington, MA, USA). The image was scanned with ChemiDoc (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab™ 5.1 software (Bio-Rad, Hercules, CA, USA) [58].
